The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Actin is the most abundant protein in animal cells and constitutes 10-20% of the protein of many nucleated cells and 30% of the protein of muscle cells. Actin molecules each bind an ATP molecule and self-assemble into long filaments during which the ATP is hydrolyzed into ADP.
Injury to animal tissues results in the release of actin into the extracellular space, including the bloodstream. Although approximately half of nonmuscle cell actin is F-actin, (the double-helical, rodlike, filament form of actin which is assembled from G-actin monomers), the ionic conditions of extracellular fluids favor actin polymerization, so that virtually all the actin released into the blood from dying cells would be expected to polymerize into filaments (Lind, S. E. et al., Am. Rev. Respir. Dis. 138:429-434 (1988)). In purified solutions, in the absence of filament-shortening proteins, actin filaments can easily attain lengths of several microns. Were some fraction of actin released from injured cells to be irreversibly denatured, however, or else bound to one of the intracellular actin-binding proteins discussed below, this actin would remain monomeric.
There are many proteins which naturally associate with actin (for a review of actin-binding proteins, see Stossel et al., Ann. Rev. Cell Biol. 1: 353-402 (1985); Pollard et al., Ann. Rev. Biochem. 55:987-1035 (1986)). However, two proteins, gelsolin and DBP (vitamin D binding protein) are thought to be primarily responsible for binding extracellular actin. (Janmey et al., Blood 70:529-530 (1987)). Gelsolin is an actin-binding protein that is a key regulator of actin filament assembly and disassembly. Gelsolin is an 82-kDa protein with six homologous subdomains, referred to as S1-S6. Each subdomain is composed of a five-stranded β-sheet, flanked by two α-helices, one positioned perpendicular with respect to the strands and one positioned parallel. The N-terminal (S1-S3) forms an extended β-sheet, as does the C-terminal (S4-S6) (Kiselar et al. PNAS 100: 3942-3947 (2003)). The protein is highly conserved and highly homologous among species. Gelsolin is located intracellularly (in cytosol and mitochondria) and extracellularly (in blood plasma). Koya et al., J Biol Chem 275 (20): 15343-15349 (2000).
Gelsolin has several functions in regulating actin polymerization. First, gelsolin is involved in monomeric actin binding. In the presence of Ca2+, gelsolin binds two actin monomers. Gelsolin can also bind actin filaments by a another actin binding site. Second, gelsolin binds two actin monomers to form a nucleus for actin polymerization and caps the barbed end of actin filaments. Thus, gelsolin is capable of both serving as a nucleus for actin polymerization and capping the ends of the nascent microfilaments. Finally, gelsolin has actin severing activity.
Because of the large amounts of actin in cells, the release of actin from dying cells provides sufficient actin to have a significant affect on the microenvironment, either by increasing the viscosity of extracellular fluids of plasma and/or by entrapping cells or by other, as yet unidentified toxic effects. Infusion of extracellular free actin is toxic to animal tissues, and especially to renal and cardiopulmonary systems (Harper et al., Clin. Res. 36:625 A (1988); Haddad et al., PNAS 87: 1381-1385 (1990)). Acute renal failure is a complication of muscle injury and actin infusions in rats causes transient elevations of the blood urea nitrogen (BUN) and creatinine levels, consistent with renal failure. Free actin in the plasma may form filaments which may lead to multiple organ dysfunction syndrome (Dahl et al., Shock 12(2): 102-4 (1999)). Moreover, since each extracellular actin molecule in a filament has an ADP molecule associated with it, the presence of extracellular actin in the blood may tend to induce or augment platelet aggregation in a manner which may not be advantageous to the host (Lind et al., Am. Rev. Respir. Dis. 138:429-434 (1988); Scarborough et al., Biochem. Biophy. Res. Commun. 100:1314-1319 (1981)). Consequently, plasma gelsolin has a vital function of scavenging actin released from dead and dying cells and plasma gelsolin levels appear to be an early prognostic marker in patients experiencing trauma (Mounzer et al., Am. J. Respir. Crit. Care Med. 160: 1673-81 (1999)).